Jack Zhu

Archival Fine-Needle Aspiration Cytopathology (FNAC) Samples: Untapped Resource for Clinical Molecular Profiling

Microarray technologies provide high-resolution maps of chromosome imbalances and epigenomic aberrations in the cancer cell genome. Such assays are often sensitive to sample DNA integrity, voiding the utility of many archival pathology specimens and necessitating the special handling of prospective clinical specimens. We have identified the remarkable preservation of higher-molecular weight DNA in archival fine-needle aspiration cytopathology specimens from patients greater than 10 years of age. We further demonstrate the outstanding technical performance of 57 fine-needle aspiration cytopathology samples for aberration detection on high-resolution comparative genomic hybridization array, DNA methylation, and single nucleotide polymorphism genotyping platforms. Forty-four of 46 malignant aspirates in this study manifested unequivocal genomic aberrations. Importantly, matched Papanicolaou and Diff-Quik fine-needle aspiration cytopathology samples showed critical differences in DNA preservation and DNA integrity. Overall, this study identifies a largely untapped reserve of human pathology specimens for molecular profiling studies, with ramifications for the prospective collection of clinical biospecimens.

Characterization of the metastatic phenotype of a panel of established osteosarcoma cells

Osteosarcoma (OS) is the most common bone tumor in pediatric patients. Metastasis is a major cause of mortality and morbidity. The rarity of this disease coupled with the challenges of drug development for metastatic cancers have slowed the delivery of improvements in long-term outcomes for these patients. In this study, we collected 18 OS cell lines, confirmed their expression of bone markers and complex karyotypes, and characterized their in vivo tumorgenicity and metastatic potential. Since prior reports included conflicting descriptions of the metastatic and in vivo phenotypes of these models, there was a need for a comparative assessment of metastatic phenotypes using identical procedures in the hands of a single investigative group. We expect that this single characterization will accelerate the study of this metastatic cancer. Using these models we evaluated the expression of six previously reported metastasis-related OS genes. Ezrin was the only gene consistently differentially expressed in all the pairs of high/low metatstatic OS cells. We then used a subtractive gene expression approach of the high and low human metastatic cells to identify novel genes that may be involved in OS metastasis. PHLDA1 (pleckstrin homology-like domain, family A) was identified as one of the genes more highly expressed in the high metastatic compared to low metastatic cells. Knocking down PHLDA1 with siRNA or shRNA resulted in down regulation of the activities of MAPKs (ERK1/2), c-Jun N-terminal kinases (JNK), and p38 mitogen-activated protein kinases (MAPKs). Reducing the expression of PHLDA1 also delayed OS metastasis progression in mouse xenograft models.

Novel near-diploid ovarian cancer cell line derived from a highly aneuploid metastatic ovarian tumor

A new ovarian near-diploid cell line, OVDM1, was derived from a highly aneuploid serous ovarian metastatic adenocarcinoma. A metastatic tumor was obtained from a 47-year-old Ashkenazi Jewish patient three years after the first surgery removed the primary tumor, both ovaries, and the remaining reproductive organs. OVDM1 was characterized by cell morphology, genotyping, tumorigenic assay, mycoplasma testing, spectral karyotyping (SKY), and molecular profiling of the whole genome by aCGH and gene expression microarray. Targeted sequencing of a panel of cancer-related genes was also performed. Hierarchical clustering of gene expression data clearly confirmed the ovarian origin of the cell line. OVDM1 has a near-diploid karyotype with a low-level aneuploidy, but samples of the original metastatic tumor were grossly aneuploid. A number of single nucleotide variations (SNVs)/mutations were detected in OVDM1 and the metastatic tumor samples. Some of them were cancer-related according to COSMIC and HGMD databases (no founder mutations in BRCA1 and BRCA2 have been found). A large number of focal copy number alterations (FCNAs) were detected, including homozygous deletions (HDs) targeting WWOX and GATA4. Progression of OVDM1 from early to late passages was accompanied by preservation of the near-diploid status, acquisition of only few additional large chromosomal rearrangements and more than 100 new small FCNAs. Most of newly acquired FCNAs seem to be related to localized but massive DNA fragmentation (chromothripsis-like rearrangements). Newly developed near-diploid OVDM1 cell line offers an opportunity to evaluate tumorigenesis pathways/events in a minor clone of metastatic ovarian adenocarcinoma as well as mechanisms of chromothripsis.

Somatic VHL Mutation in a Patient With MEN1-Associated Metastatic Pancreatic Neuroendocrine Tumor Responding to Sunitinib Treatment: A Case Report

Multiple endocrine neoplasia type 1 (MEN1) and von Hippel-Lindau (VHL) are autosomal-dominant diseases caused by germline mutations in tumor-suppressor genes. A patient with a germline MEN1 mutation and a somatic VHL mutation in the tumor has not been reported. Herein, we report on a patient with MEN1 and a metastatic nonfunctioning pancreatic neuroendocrine tumor (PNET) with a somatic VHL mutation. This patient underwent a pancreaticoduodenectomy for a grade 2 PNET obstructing her pancreatic duct. The patient developed liver and regional lymph node metastases as well as growth of a PNET in the remnant pancreas. As part of a clinical trial for mutation-targeted therapy, a biopsy of the metastatic tumor was obtained. The clinical diagnosis, confirmed by OncoVAR-NET and molecular profiling analysis, revealed MEN1 with a germline deletion in exon 2 and a c.402 deletion C, p.Phe134LeufsX51. In addition, a somatic mutation in the VHL gene—a nonsense mutation, c.529A>T, p.Arg177Ter—was identified by hybrid capture sequencing. The mutations were confirmed by Sanger sequencing. Comparative genomic hybridization showed loss of heterozygosity in both the MEN1 and VHL genes. The patient was treated with sunitinib and had a partial response to treatment. This case illustrates not only that a second hit occurs in tumor suppressor genes but that somatic mutations are also possible in additional tumor suppressor genes. This suggests that targeted therapy selection should include analysis of somatic mutations even when the susceptibility gene is known.

Abstract 1438: The dynamics of genetic aberrations in Crohn's disease associated colorectal carcinogenesis

Crohn’s disease, a condition of chronic inflammation of the intestine, significantly increases the risk for the development of colorectal cancer (CRC). Sporadic CRCs are characterized by a specific pattern of genomic imbalances and a landscape of acquired gene mutations. In this study we aimed to compare CRCs that arise as a consequence of chronic inflammation in Crohn’s disease with sporadic CRCs. We analyzed 26 Crohn’s disease associated CRCs, four matched dysplastic lesions, six matched inflamed mucosa samples, and two matched lymph node metastases using array comparative genomic hybridization, targeted sequencing (564 cancer related genes) and gene expression profiling. As a control, we used normal intestinal mucosa from the resection margin of these CRCs and 24 sporadic CRCs. In general, CRCs that developed in patients with Crohn’s disease had a similar distribution of genomic imbalances compared to sporadic CRC. However, we identified distinct mutation signatures: in the Crohn9s disease associated CRCs the most frequently mutated gene was TP53, which occurred in 65% of the samples. The second, third and fourth most frequently mutated genes were KRAS (27%), APC (23%) and PIK3CA (19%). In the control group of sporadically arising CRCs, the most commonly mutated gene was APC (75%), followed by KRAS (54%), TP53 (33%), SMAD3 (29%) and SMAD2 (25%). The genetic analyses of multiple lesions from individual patients revealed a high degree of intertumoral heterogeneity with diverse patterns of gene mutations and allowed the reconstruction of the sequence of genetic events during Crohn’s disease associated tumorigenesis. In contrast to sporadic colorectal carcinogenesis, TP53 mutations were observed as early and common events while APC mutations occurred rather late and were infrequent. Our comprehensive molecular profiling of Crohn’s disease associated CRCs suggests that the genetic landscape of CRC is influenced by the activation of inflammation related pathways. Furthermore, our findings offer potential for establishing an early detection marker for dysplasia in patients with Crohn’s disease. Citation Format: Daniela D. Hirsch, Darawalee Wangsa, Yue Hu, Jack Zhu, David Petersen, Daniel C. Edelman, Paul S. Meltzer, Bao Tran, Kerstin Heselmeyer-Haddad, Thomas Ried, Timo Gaiser. The dynamics of genetic aberrations in Crohn9s disease associated colorectal carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1438. doi:10.1158/1538-7445.AM2017-1438

Abstract 3851:NUP98-PHF23(NP23) binds H3K4me3 and causes a wide spectrum of leukemias, including AML, T-ALL, and pre-B1 ALL.

Nucleoporin 98 (NUP98) fusions have been associated with acute myeloid leukemia (AML), chronic myeloid leukemia, myelodysplastic syndrome, and T-cell acute lymphoblastic leukemia (T-ALL). An AML patient with a t(11;17)(p15;p13) chromosome translocation was found to have a novel gene fusion between NUP98 and plant homeodomain (PHD) finger 23 (PHF23). PHF23 has been reported to act as a reader of tri-methylated histone 3 lysine 4 (H3K4me3) marks, suggesting that PHF23 may function in chromatin regulation and that the NP23 fusion protein may play a role in aberrant chromatin modification. To determine the oncogenic potential of NP23, we generated mice that expressed the NP23 fusion in hematopoietic tissues. We characterized two founder lines (C10 and B10) that expressed the NP23 fusion. The C10 line principally developed AML that closely resembled the human disease, with increased blasts in the blood and bone marrow, widespread organ infiltration, and myeloid immunophenotype. Onset of disease was as early as 4.5 months, and 70 percent of the NP23 mice succumbed to leukemia by 12 months of age. The B10 line developed a wider spectrum of leukemias with similar age of onset and penetrance. The B10 mice predominantly developed T-ALL and AML. Interestingly, four B10 mice developed a previously unreported form of ALL that was of pre-B1 (B220−/CD19+/AA4.1+) origin, indicating that the NP23 protein is oncogenic in multiple hematopoietic lineages. Gene expression arrays and RQ-PCR revealed Hoxa5, a7, a9 and a10 to be overexpressed from 10-1000 fold in both AML and T-ALL, as well as in hematopoietic tissues from clinically healthy NP23 transgenic mice. Premalignant, clinically healthy mice showed aberrant hematopoietic stem and progenitor cell (HSPC) populations. Bone marrow lineage negative (Linneg) cells were decreased by half that of WT littermates, and LSK (Linneg/Sca1+/cKit+) cells were further reduced by 3-fold. NP23 thymi were dramatically smaller in size with an average 5-fold reduction in thymocytes, which was associated with a partial differentiation block of thymocytes at the DN2 stage. B2 pre-B-cells were significantly reduced, by 50%, compared to WT controls. B1-progenitor B-cells (Linneg/CD19+/AA4.1+) in the bone marrow were reduced by 30% in the bone marrow and mature B1 B-cells (B220−/CD19+/IgM+) of the peritoneal cavity were further decreased to half that of WT. We also performed genome wide NP23 and H3K4me3 ChIP-seq which identified a core set of H3K4me3 marked genes bound by the NP23 fusion protein that were overexpressed in both myeloid and T-cell lineages. The NP23 oncogene is capable of promoting cell lineage-independent leukemic transformation, and will be useful in identifying oncoproteins and transformation pathways that involve chromatin deregulation and HSPC gene signatures such as those of the HOXA cluster. Citation Format: Sheryl M. Gough, Fan Lee, Yang Jo Chung, Robert L. Walker, Fan Yang, Jack Zhu, Yi Ning, Paul S. Meltzer, Peter D. Aplan. NUP98-PHF23 (NP23) binds H3K4me3 and causes a wide spectrum of leukemias, including AML, T-ALL, and pre-B1 ALL. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3851. doi:10.1158/1538-7445.AM2013-3851

Abstract 1493: High-resolution comparative genomic hybridization of preneoplastic and invasive esophageal squamous cell carcinoma: Identification of the earliest preneoplastic changes

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Background: Esophageal cancer causes > 400,000 deaths/year in the world. Clinically useful early detection methods must identify these cancers and their precursor lesions at more curable stages. We conducted comparative genomic hybridization analyses of preneoplastic and invasive esophageal squamous cell carcinoma to look for gene copy number patterns that might be useful as early detection markers. The current effort focuses on aberrations associated with mild squamous dysplasia, the earliest histologic form of squamous preneoplasia. Methods: DNA was extracted from either biopsies or surgical resections from 122 esophageal tissues spanning a spectrum including normal mucosa, squamous preneoplasia, and invasive squamous cell carcinoma. High-resolution micro-array based comparative genomic hybridization (Agilent) was used to measure regions of chromosomal gain or loss with an emphasis on identifying changes associated with the transition from normal to mild dysplasia. Results: Chromosomal number aberrations were identified that involve several chromosomes, occur early in the neoplastic process, and progress with disease severity. Specific chromosomal number aberrations associated with mild squamous dysplasia, the earliest form of squamous preneoplasia, were uniquely identified. Conclusions: Comparative genomic hybridization identifies patterns of chromosomal number aberrations that may distinguish patients with and without high-grade squamous dysplasia or cancer. The current effort also identifies specific changes associated with mild squamous dysplasia, the earliest form of preneoplasia, and this may help elucidate mechanisms related to the initiation of carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1493.

Abstract 4612: Analysis of transcriptional regulation by progesterone receptor in ER-positive breast cancer cells by ChIP-Seq

Breast cancer is among the most common malignancies in women. It is well established that progesterone receptor (PR) is a major determinant in the development and progression of breast tumor, as well as in diagnosis and treatment of breast cancer. However, it is still not fully understood how PR affects downstream signaling pathways and cross talks with other intracellular signal transduction pathways. To better define the role played by PR in estrogen receptor (ER)-dependent gene regulation, we have used chromatin immunoprecipitation coupled with next generation sequencing (ChIP-Seq) to examine the in vivo DNA binding by PR. Whole-Genome ChIP-Seq analysis has identified large number of PR binding sites in T-47D breast cancer cells, which are ER-positive and PR-positive. PR binding sites were detected in proximal promoter regions as well as distal regions, with most located in non-proximal regions. The comparison of PR and ER binding sites revealed that a significant of portion of the PR binding sites overlaps with the ER binding sites, indicating a close relationship between PR and ER in ER-dependent gene regulation. The PR motifs were found to be among the most enriched sequence motifs within the PR binding sites. Further analysis using the chromosome conformation capture assay revealed that progesterone induces changes to the chromatin structures of known PR-regulated genes. The ChIP-Seq data, together with the expression profiling and other results, indicate that PR may cooperate with ER and play an important role in ER-dependent gene regulation, mammary epithelial differentiation and breast cancer development. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4612.